1402
T. Sasami et al. / Bioorg. Med. Chem. 17 (2009) 1398–1403
tetrazole (69 mg, 0.98 mmol) and 2-cyanoethyl N,N,N0,N0-tetraiso-
propylphosphorodiamidite (678 l, 2.1 mmol). The solution was
4. Experimental procedures
4.1. General procedures
l
stirred at ambient temperature for 2 h, and then dissolved in ethyl
acetate (150 ml), washed three times with NaHCO3 (150 ml). The
organic layer was dried over MgSO4, filtered and concentrated un-
der reduced pressure. The residue was chromatographed on a silica
gel column with chloroform–methanol containing 0.5% triethyl-
amine (97:3–96:4) to give 2 (1.2 g, 90%). 1H NMR (CDCl3-d1)
1.05–1.32 (14H, m), 2.42 (1H, m), 2.56–2.67 (3H, m), 2.91 (4H,
m), 3.25–3.37 (2H, m), 3.51–3.87 (8H, m), 4.20–4.23 (1H, m),
4.69 (1H, m), 5.81 (2H, br), 6.32 (1H, m), 6.70–6.78 (4H, m),
7.12–7.37 (9H, m), 7.80 (1H, m), 8.89 (1H, br) 10.00–12.00 (1H,
br): 13C NMR (CDCl3-d1) 18.6, 19.5, 20.4, 20.5, 20.6, 20.8, 23.3,
24.7, 24.8, 29.5, 29.9, 40.4, 43.5, 45.7, 47.1, 55.4, 55.5, 55.5, 58.3,
58.5, 58.6, 63.6, 63.7, 83.7, 85.6, 85.8, 86.7, 86.7, 113.2, 113.4,
117.9, 118.1, 120.0, 120.1, 127.2, 128.1, 128.4, 128.5, 130.3,
135.8, 135.8, 135.8, 135.9, 136.0, 144.7, 149.2, 156.8, 158.7,
158.8: 31P NMR (CDCl3-d1) 149.9: MS m/z Calcd for C41H50N8O8P+:
813.3489, found 813.3712.
The dry solvents were purchased and stored over molecular
sieves 4A. 1H, 13C and 31P NMR spectra were obtained at 500,
126 and 203 MHz, respectively. The chemical shifts were measured
from tetramethylsilane (0.0 ppm) or DMSO-d6 (2.49 ppm) for 1H
NMR, CDCl3 (77.0 ppm), DMSO-d6 (39.7 ppm) for 13C NMR and
85% phosphoric acid (0.0 ppm) for 31P NMR. MALDI-TOF and ESI-
TOF mass spectra were obtained in the positive ion mode.
4.2. Synthesis of phosphoramidite unit
4.2.1. 2-N-Carbamoyl deoxyguanosine (4)
Deoxyguanosine 3 (1.0 g, 3.5 mmol) was rendered anhydrous
by repeated coevaporation with dry pyridine. The residue was
suspended in dry pyridine (30 ml) was added trimethylsilyl chlo-
ride (1.8 ml, 21 mmol). The solution was stirred at ambient tem-
perature 1 h, and then added phenyl chloroformate (660 ll,
4.3. 30,50-O-Bis(tert-butyldimethylsilyl)-2-N-phoxyacetyl-
deoxyguanosine (7)
5.3 mmol). The solution was stirred at ambient temperature 5 h.
The solution was diluted with ethyl acetate (150 ml) and washed
twice with brine (150 ml). The organic layer was dried over
MgSO4, filtered and concentrated under reduced pressure. The
residue was diluted with pyridine and added aqueous ammonia
(5.0 ml, 35 mmol). The solution was concentrated under reduced
pressure. The residue was purified by the precipitation from
methanol (5 ml) to give the compound 4 (600 mg, 55%). 1H
NMR (DMSO-d6) 2.22–2.27 (1H, m), 2.52–2.57 (1H, m), 3.49–
3.56 (2H, m), 3.80 (1H, dd, J = 4.6 Hz, J = 7.6 Hz), 4.35 (1H, t,
J = 2.6 Hz), 4.92 (1H, s), 5.29 (1H, s), 6.15 (1H, t, J = 6.7 Hz), 6.44
(1H, s br), 7.23 (1H, s br), 8.12 (1H, s), 10.01 (1H, s br), 11.99
30,50-O-Bis(tert-butyldimethylsilyl)-2-N-carbamoyldeoxyguano-
sine 6 (200 mg, 0.37 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine. The residue was suspended in
dry pyridine (3.7 ml). To this mixture was added phenoxyacetic
anhydride (425 mg, 1.5 mmol), and the mixture was stirred at ambi-
enttemperaturefor 24 h. Thesolutionwas dilutedwith ethyl acetate
(50 ml) and washed twice with brine (50 ml) and once with satu-
ratedNaHCO3aq (50 ml). TheorganiclayerwasdriedoverMgSO4, fil-
tered and concentrated under reduced pressure. The residue was
chromatographed on a silica gel column with hexane–ethyl acetate
(70:30) to give mixture of 6 and phenoxyacetamide (180 mg, 69%,
molar ration of 6 and phenoxyacetylamide was 2:1). 1H NMR
(DMSO-d6) 0.03 (6H, s), 0.10 (6H, s), 0.86 (9H, s), 0.88 (9H, s), 2.29–
2.34 (1H, m), 2.69–2.75 (1H, m), 3.31–3.73 (2H, m), 3.84 (1H, d,
J = 6.3 Hz), 4.51 (1H, d, J = 2.3 Hz), 4.84 (2H, s), 6.20 (1H, d,
J = 6.6 Hz), 6.93–6.99 (3H, m), 7.27–7.32 (2H, m), 8.21 (1H, s),
11.78 (1H, br): 13C NMR (DMSO-d6) ꢀ5.5, ꢀ5.5, ꢀ5.0, ꢀ4.8 17.7,
18.0, 25.7, 25.9, 62.6, 66.3, 72.0, 82.7, 87.3, 114.6, 120.5, 121.4,
129.6, 137.4, 147.3, 148.3, 154.9, 157.6, 170.9: MS m/z Calcd for
C30H48N5O6Si2+: 630.3143, found 630.3193.
(1H, s br): 13C NMR (DMSO-d6) 61.5, 70.5, 83.1, 87.7, 119.3,
+
137.0, 148.8, 148.9, 155.2, 156.2: MS m/z Calcd for C11H15N6O5
:
311.1104, found 311.1088.
4.2.2. 2-N-Carbamoyl-50-O-(4,40-dimethoxytrityl)
deoxyguanosine (5)
2-N-Carbamoyldeoxyguanosine 4 (1.4 g, 4.6 mmol) was ren-
dered anhydrous by repeated coevaporation with dry pyridine.
The residue was suspended in dry DMF (35 ml). To this mixture
was added pyridine (1.9 ml, 23 mmol) and 4,40-dimethoxytrityl
chloride (2.4 g, 7.0 mmol), and the mixture was stirred at ambient
temperature for 12 h. The solution was diluted with ethyl acetate
(250 ml) and washed twice with brine (200 ml) and once with sat-
urated NaHCO3aq (200 ml). The organic layer was dried over
MgSO4, filtered and concentrated under reduced pressure. The res-
idue was purified by the precipitation from ethyl acetate (5 ml)
and n-hexane (5 ml) to give the compound 5 (2.4 g, 84%). 1H
NMR (DMSO-d6) 2.31–2.35 (1H, m), 2.66–2.71 (1H, m), 3.10–3.17
(2H, m), 3.71 (6H, d, J = 1.7 Hz), 3.92 (1H, m), 4.39 (1H, dd,
J = 5.3 Hz, J = 10.5 Hz), 5.33 (1H, s br), 6.20 (1H, t, J = 6.2 Hz),
6.77–6.82 (4H, m), 7.16–7.38 (9H, m), 7.99 (1H, s): 13C NMR
(DMSO-d6) 55.0, 55.0, 64.0, 70.3, 83.0, 85.4, 85.8, 113.0, 113.1,
119.5, 126.6, 127.4, 127.7, 129.7, 129.1, 129.7, 135.5, 135.6,
136.9, 144.9, 148.8, 148.9, 156.2, 158.0, 158.0: MS m/z Calcd for
C32H33N6NaO7+: 635.2230, found 635.2229.
4.4. Oligonucleotide synthesis
The ODNs incorporating a 2-N-carbamoyl deoxyguanosine was
synthesized on an Applied Biosystems 394 automated DNA/RNA
synthesizer using the standard 1.0 mol phosphoramidite cycle of
detritylation, coupling, capping and iodine oxidation. The phos-
phoramidites 1 and 2 were dissolved in dry acetonitrile so that
the final concentration became 0.1 M. The oligonucleotides were
cleaved from the CPG supports and the protecting groups were re-
moved by treatment with aqueous ammonia for 8 h. The solution
containing the DMTr-ON oligonucleotides thus obtained were
placed on the C 18 cartridge column and the failure sequences
were eluted by use of 10% CH3CN/0.1 M ammonium acetate as an
eluent. After being washed with 0.1 M ammonium acetate and
water, the column was treated with aqueous 2% TFA to remove
DMTr group, washed with 0.1 M ammonium acetate and water.
The target oligonucleotide was eluted by use of 20% CH3CN/water.
Pure material was obtained by use of the C18 cartridge column to
give the pure oligonucleotide after being lyophilized to dryness.
The yield of the pure material was calculated by assuming the mo-
lar extinction coefficients. The structures were confirmed by MAL-
DI-TOF mass spectroscopy.
4.2.3. 2-N-Carbamoyl-50-O-(4,40-dimethoxytrityl)-
deoxyguanosine 30-(2-cyanoethyl-N,N-
diisopropylphosphoramidite) (2)
Compound 5 (1.0 g, 1.6 mmol) was rendered anhydrous by
coevaporation three times with dry pyridine and toluene and ace-
tonitrile and finally dissolved in dry CH2Cl2 (16 ml). To this solu-
tion were added diisopropylamine (137 ll, 0.98 mmol), 1H-